We have continued to develop selective biophysical measurement systems (biological atomic force microscopy (Bio-AFM) platforms, Quartz Crystal Microbalance-Dissipation (QCM-D), and optical microscopy and spectroscopy), and to apply these technologies to important biomedical investigations in collaboration with outstanding NIH intramural and extramural scientists. We are working toward broader and more insightful applications of multifunctional, multimodal, and multiplatform AFM imaging and single molecule force spectroscopy (SMFS) for cellular and macromolecular studies. On biomedical applications, we have continued our broad range of collaborations that include: (1) We have advanced our commitment to developing a better clinical vaccine toward enhanced immunological response and eventual eradication of malaria. Over several years and via Bio-AFM and related bioanalysis, we have investigated the macromolecular structure and nanomechanical properties of more and malaria vaccine candidates and virus-like-particle (VLP) or liposome carriers with Dr. David Narum (NIAID, NIH) and other collaborators. These malaria protein antigens and vaccine carriers are produced via recombinant-protein biotechnology, purified, and characterized in a manner suitable for human trials and scale-up production. Biophysical characterization at single macromolecule and assembly level using Bio-AFM imaging and force spectroscopy are helping to define these vaccine constructs along the developmental phases. In this year, we worked on extracting and characterizing Plasmodium falciparum lipid rafts and GPI-anchored proteins to improve mechanistic understanding of the malaria parasites and pathogen-host interactions. This work follows upon our published observation of a reversible conformation change and adhesion domain masking in the Plasmodium falciparum circumsporozoite protein (CSP), the leading malaria vaccine target. (2) We have expanded our collaborations on multifunctional nanomedicine and theranostics with Dr. Xiaoyuan Chen (laboratory of Molecular Imaging and Nanomedicine, NIBIB) and co-investigators including Dr. Peng Huang (NIBIB) and Dr. Zhe Wang (NIBIB). Focusing on understanding and fighting cancer, we have contributed to several published and ongoing studies such as using tumor-specific formation of enzyme-instructed supramolecular assemblies as cancer theranostics. We are applying Bio-AFM and QCM-D methodology to investigate nanoparticle theranostics, cancer cells and stem cells, and other biomedical systems. (3) We have continued our multi-year Bio-AFM studies of protein clathrin and assemblies with collaborators including Prof. Eileen Lafer and Prof. Rui Sousa (Univ. Texas Health Sciences Center, San Antonio), Dr. Ralph Nossal (NICHD) and Dr. Dan Sackett (NICHD)in the key area of the receptor-mediated endocytosis and intracellular trafficking. This collaboration has expanded toward bio-AFM studies of microtubules interacting with several approved or developing anti-cancer drugs. Related to exocytosis and endocytosis, we are collaborating also with Dr. Ling-gang Wu (NINDS) and coworkers to better understand synaptic transmission and neuronal communications in brain. (4) We have collaborated with Dr. Andrew Doyle and Dr. Kenneth Yamada (Laboratory of Cell and Developmental Biology, NIDCR) and with Dr. Raimon Sunyer (Institute for Bioengineering of Catalonia, Spain) on nanomechanics and structural properties of reconstituted extra cellular matrix (ECM)-like collagen gels via Bio-AFM force spectroscopy to investigate cell adhesion, migration, and other dynamic behaviors. We have refined force spectroscopy and quantitative nanomechnical mapping (QNM) approaches to explore more completely tissue-mimicking soft material and tissue specific extracellular matrices. (5) With Dr. Curtis Meuse(NIST) and coworkers, we have advanced our Bio-AFM and biophysical studies of amyloid-beta fibrils in the Alzheimer's disease, especially to resolve assembly pathways in physiologically relevant fluid and surface environments. We advanced on a project with Prof. Qi Lu (Delaware State University) and co-investigators, on the effect of nanoparticles on lipid domains and membrane properties. Finally, we are combining AFM and optical microscopy in other continuing and new collaborations, including biomembranes, protein assemblies, DNA and protein crosslinking, DNA-protein interactions and cell cycles, that are critically important to disease mechanisms and bionanotechnology.